DNA minicircles are nano-objects of great interest both for basic research studies and for multiple applications in nanotechnology and nucleic acids medicine.
DNA minicircles, either double- or single-stranded, can be used as nanoscale building objects in structural DNA nanotechnology, a growing field of research. For example, DNA minicircles have been used as scaffold to assemble nanoarchitectures after the incorporation of G-quadruplexes, RNA hairpin or chemically functionalized oligonucleotide leading to the possibility to create molecular devices with a broad range of functions. The ability to create a controlled bend in a structural material is an important feature that has been exploited for nanomachine building. This biological application of DNA nanostructures represents a very young field of research that necessitates amounts of DNA minicircles building material.
DNA minicircles have also promising applications as new biologically active nucleic acid molecule in gene therapy approaches. In particular DNA minicircles can be used as inhibitors of specific gene expression, because they offer the exciting possibility to block the expression of critical genes through the inhibition of key players in transcription regulation (i.e. transcription factors) without any changes in functions of other genes. In particular, the delivery of circular nucleic acids in biological fluid is also known to present several advantages as compared to linear oilgonucleotides such as an increased stability thanks to resistance to exonucleases. It is also well recognized that reducing the size of DNA is beneficial in gene therapy approaches by improving cell DNA transfection and trafficking (Lukacs, G. L et al., 2000, J. Biol. Chem. 275, 1625-1629; Kreiss, P. et al., 1999 Nucleic Acids Res. 27, 3792-3798).
Furthermore, DNA supercoiling is a relevant structural characteristic in DNA-dependent cellular processes (Kanaar, R. and Cozzarelli, N. R., 1992 Curr. Opin. Struct. Biol. 2, 369-379; Baranello, L. et al., 2012, Biochim. Biophys. Acta 1819, 632-638). DNA minicircles present the advantage to mimics the DNA loops that are formed during essential DNA-dependent transactions, such as transcription, replication and recombination. Therefore the use of supercoiled minicircles may help investigate higher order DNA structure of biological relevance on the binding and activity of proteins implicated in the DNA metabolism (transcription factors and repair proteins for instance).
The use of plasmids designed for in vivo site specific recombination allows production of minicircle containing supercoiling (Fogg, J. M. et al., 2006, J. Phys. Condens. Matter 18, S145-S159) in milligram quantities for DNA, however, the direct production of chemically functionalized minicircles is impossible as a consequence to in cellulo minicircle production. Furthermore, the versatility of this approach for introducing customized DNA sequences is limited by the labor intensive method and the likelihood of sequence-dependent unpredictable negative effect on the recombination efficiency and the production of short DNA fragment smaller than 250 bp is inefficient. Heine et al. have described (in a poster retrievable from the internet: URL: http://rentschler.de/fileadmin/Downloads/Poster/Rentschler-Poster-In-Vitro-2011_Handout.pdf), a semi-synthetic method for the production of DNA minicircles, wherein linear substrates, obtained after plasmid digestion by restriction enzymes and a purification step, are circularized using a ligase enzyme to produce relaxed circular DNA. A DNA gyrase can be used after the circularization step for inducing DNA. However, this method does not describe the production of minicircles down to 250 base pairs.
Indeed, the construction of minicircles from short linear DNA fragments down to 250 base pairs (bp) and containing random sequences remains difficult because such DNA length is in the vicinity of the persistent length (about 150 bp/50 nm, the persistence length being a measure to characterize the stiffness regarding intrinsic DNA flexibility). As a consequence the yield of the monomolecular reaction, i.e. the circularization of DNA by closure of both DNA ends by enzymatic ligation, is weak as compared to that of the competitive bi- and multi-molecular reactions between DNA double helix even at low range of DNA concentration (less than 1 nM). Consequently, only a few in vitro methods have indeed been reported for ligase-mediated circularization of linear DNA fragments of less than 250 pb.
In the absence of bending proteins, a slight increase in the efficiency of the cyclization reaction has been achieved using specific sequences endowed with intrinsic bendability (so called adenine tracts) with the drawback to lose the possibility to incorporate freely DNA sequence of interest, such as consensus sequence for protein recognition (Zhang, Y. and Crothers, D. M., 2003, Proc. Nat. Acad. Sci. USA 100, 3161-3166). Another in vitro strategy has employed DNA substrates with very long cohesive ends allowing hybridization of their single-stranded region, to increase the overall yield of minicircle formation but a low DNA concentration used in the cyclization reaction and labor intensive preparation steps remain important drawbacks for minicircle quantitative production (Du, Q. et al., 2008, Nucleic Acids Res. 36, 1120-1128).
In the presence of a DNA bending protein, random-sequence DNA fragments have been used in DNA ligase-dependent circularization reactions to determine the ability of various architectural proteins to induce DNA bending/flexibility. However, the nanomolar concentration range of linear DNA substrate is a limitation towards the possibility of minicircle production.
Lastly, to the applicant knowledge, none of the above-mentioned cell-free methods showed the possibility to yield supercoiled small DNA minicircles.
Therefore there is still a need for a method allowing quantitative and efficient production of DNA minicircles, of length down to about 250 base pairs with controlled supercoiling and customized DNA sequences.